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Computationally-predicted CB1 cannabinoid receptor mutants show distinct patterns of salt-bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding

Ahn, Kwang H. and Scott, Caitlin E. and Abrol, Ravinder and Goddard, William A., III and Kendall, Debra A. (2013) Computationally-predicted CB1 cannabinoid receptor mutants show distinct patterns of salt-bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding. Proteins, 81 (8). pp. 1304-1317. ISSN 0887-3585. PMCID PMC4872635. https://resolver.caltech.edu/CaltechAUTHORS:20140124-094525878

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Abstract

The cannabinoid receptor 1 (CB1), a member of the class A G-protein-coupled receptor (GPCR) family, possesses an observable level of constitutive activity. Its activation mechanism, however, has yet to be elucidated. Previously we discovered dramatic changes in CB1 activity due to single mutations; T3.46A, which made the receptor inactive, and T3.46I and L3.43A, which made it essentially fully constitutively active. Our subsequent prediction of the structures of these mutant receptors indicated that these changes in activity are explained in terms of the pattern of salt-bridges in the receptor region involving transmembrane domains 2, 3, 5, and 6. Here we identified key salt-bridges, R2.37 + D6.30 and D2.63 + K3.28, critical for CB1 inactive and active states, respectively, and generated new mutant receptors that we predicted would change CB1 activity by either precluding or promoting these interactions. We find that breaking the R2.37 + D6.30 salt-bridge resulted in substantial increase in G-protein coupling activity and reduced thermal stability relative to the wild-type reflecting the changes in constitutive activity from inactive to active. In contrast, breaking the D2.63 + K3.28 salt-bridge produced the opposite profile suggesting this interaction is critical for the receptor activation. Thus, we demonstrate an excellent correlation with the predicted pattern of key salt-bridges and experimental levels of activity and conformational flexibility. These results are also consistent with the extended ternary complex model with respect to shifts in agonist and inverse agonist affinity and provide a powerful framework for understanding the molecular basis for the multiple stages of CB1 activation and that of other GPCRs in general.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1002/prot.24264 DOIArticle
http://onlinelibrary.wiley.com/doi/10.1002/prot.24264/abstractPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872635PubMed CentralArticle
ORCID:
AuthorORCID
Abrol, Ravinder0000-0001-7333-6793
Goddard, William A., III0000-0003-0097-5716
Additional Information:© 2013 Wiley Periodicals, Inc. Received 17 September 2012; Revised 21 December 2013; Accepted 21 January 2013; Published online 14 February 2013. Grant sponsor: National Institutes of Health; Grant number: DA020763 (to D.A.K), NS071112, NS073115, AI040567 (to W.A.G.)
Funders:
Funding AgencyGrant Number
NIHDA020763
NIHNS071112
NIHNS073115
NIHAI040567
Subject Keywords:GPCR; constitutive activity; protein structure prediction; ligand binding; G-protein coupling; thermal stability
Issue or Number:8
PubMed Central ID:PMC4872635
Record Number:CaltechAUTHORS:20140124-094525878
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20140124-094525878
Official Citation:Ahn, K. H., Scott, C. E., Abrol, R., Goddard, W. A. and Kendall, D. A. (2013), Computationally-predicted CB1 cannabinoid receptor mutants show distinct patterns of salt-bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding. Proteins, 81: 1304–1317. doi: 10.1002/prot.24264
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:43504
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:29 Jan 2014 00:18
Last Modified:03 Oct 2019 06:08

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